Tumor necrosis factor inhibitors

ABSTRACT

The present invention is directed to compounds that are allosteric inhibitors of tumor necrosis factor receptor I, compositions comprising such compounds, and methods of using such compounds and compositions thereof in the treatment of TNF-α mediated conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a 371 National Phase of InternationalApplication No. PCT/US06/03574, filed Jan. 31, 2006, which claims thebenefit under 35 U.S.C. 119(e), of U.S. Provisional Application60/648,973, filed Jan. 31, 2005, both of which are hereby incorporatedherein by reference in their entireties.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part in the course of research sponsored bythe National Institutes of Health grant number RO1-CA89481 and theNational Cancer Institute grant number 1PO1 CA89480-04. The U.S.government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to compounds that are allostericinhibitors of tumor necrosis factor receptor I and methods of usethereof.

BACKGROUND OF THE INVENTION

Research leading to the present invention was supported in part by fundsfrom the National Institutes of Health, the National Cancer Institute,and the Leonard and Madlyn Abramson Family Cancer Research InstituteFund.

Structural changes in proteins can be induced by various physicalfactors including pH, solvents, ligand binding and oligomerization.Conformational changes can occur at a defined local site or, as observedin multimeric proteins, at a distance from the ligand binding site(allosterism).

Protein function can be altered by conformational changes.Immunoglobulins have been shown to alter the function of proteins byinducing small to large conformational changes, and by affecting theoligomerization of proteins. For example, in the crystal structure ofTaq DNA polymerase complex, an antibody inhibited the function of DNApolymerase by inducing a large conformational change in the helix andtrapping the protein in a transition state suggesting that alteringconformational configuration at distinct sites away from the bindingsites might be used to modulate protein function.

It has been generally argued that conformational changes may be a stepin substrate/ligand recognition. Several studies from the crystalstructures of protein-protein complexes revealed conformational changesranging from 2-20 Å (0.2-2 nm) either locally or globally betweensubdomains. In the case of multimeric proteins such as myoglobin orglycogen phosphorylase, with known allosteric sites, definedconformational changes are transmitted through regions of the proteinfor regulatory or functional effects.

While surface cavities on non-enzymatic classes of proteins have beenlargely unexplored, inactivation of enzymes has been accomplished bydesigning competitive or substrate analog inhibitors that bind at activesites. Several therapeutic inhibitors have been developed based on thestructure and molecular properties of substrates and these are generallyknown as “substrate analogs”. Small molecule effectors have beenidentified for enzymes. For example, allosteric inhibitors have beendesigned and developed based on the knowledge of known and establishedallosteric binding sites. Small conformational perturbations near theactive site/ligand binding sites or polymorphisms near the active sitehave been suggested to be responsible for resistance to substrate basedinhibitors.

Tumor necrosis factor α (TNF-α) is a pleiotropic cytokine produced byactivated macrophages/monocytes and lymphocytes. TNF-α is a potentmediator in inflammatory and immune responses, including the recruitmentof leukocytes to injured tissues during bacterial and other microbialinfections, and following stimulation with inflammatory substances. Whenpresent in excessive quantities, TNF-α is known to cause tissue injury,and has been implicated in the pathology associated with inflammatoryand autoimmune diseases.

The biological effects of TNF-α, are mediated through two distinctmembrane-protein receptors, TNF-RI and TNF-RII (in humans, p55 and p75,respectively), which differ in sequence and molecular mass. TNF-RI isreported to be present at low levels in most, if not all, human celltypes, and expression of the TNF-RI gene in humans can be upregulated byinfection, interferons, and modulators of second messengers, such asphorbol esters. The extracellular portions of both TNF receptors alsoexist in soluble forms, which are derived from membrane-bound forms ofthe receptors by proteolytic cleavage at the cell surface. The solubleTNF receptors retain the ability to bind TNF-α in solution. Soluble TNFreceptors have been identified in urine and sera from healthyindividuals, and have been shown to be elevated in some chronic diseasesand following inoculation with agents that induce TNF-α release.

The pathological effects of TNF-α can be alleviated by administration ofsoluble TNF-R fragments or anti-TNF-α antibodies. These agents bindcirculating TNF-α, thus preventing the binding of TNF-α to TNF-R andlowering TNF-α signaling. TNF-R fragments or anti-TNF-α antibodies havebeen approved, by the U.S. Food and Drug Administration, for treatmentof rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosingspondylitis, psoriatic arthritis, and psoriasis.

The efficacy of TNF-R fragments and anti-TNF-α antibodies in treatingTNF-α-mediated conditions demonstrates that reducing signaling throughthe TNF-α/TNF-R signaling pathway can be used effectively to treatTNF-α-mediated conditions. TNF-R fragments and anti-TNF-α antibodies,however, are expensive to produce. Moreover, these proteinaceous agentsrequire intravenous administration.

There is, therefore, a need in the art for additional agents that reducesignaling through the TNF-α/TNF-R signaling pathway and that can be usedfor treatment of TNF-α-mediated conditions. Accordingly, the presentinventors have discovered small molecule compounds that bind to anallosteric site on TNF-R1, thus inhibiting binding of TNF-α to TNF-R1and reducing activity of the TNF-α/TNF-R1 signaling pathway. Thecompounds are useful for treatment of TNF-α mediated conditions.

SUMMARY OF THE INVENTION

The present invention is directed to compounds that are inhibitors ofTNF-R1, compositions thereof, and methods of using such compounds andcompositions to treat conditions mediated by the TNF-R1/TNF-α signalingpathway.

In certain embodiments, the invention is directed towards compoundsrepresented by formula (I), (II), or (III)

-   -   wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, and R¹²        are each independently hydrogen, alkyl, hydroxyl, alkoxy,        —NR¹³R¹⁴, halo, nitro, cyano, borono, aryl, aryloxy,        —(CH₂)_(n)COOR¹⁵, —O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷, —CR¹⁸═NOH,        —CR¹⁹R²⁰NHOH, —SO₃H, —SO₂R²¹, —SO₂NHR²², —O(CH₂)_(m)OR²³,        —C(OH)═N(OH), —C(O)NR²⁴OH, —CHR²⁵N(COR²⁶)OH, or —C(O)R²⁷ or R⁴        and R⁵ together form —NR²⁸C(O)—, —C(O)NR²⁹—, —C(O)O—, or        —S(O)₂NR³⁰—, or R⁸ and R⁹ together form —O—, —NHC(O)—, —C(O)NH—,        —C(O)O—, —NR²⁹—, or —S(O)₂NH;    -   R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵,        R²⁶, R²⁸, R²⁹, and R³⁰ are each independently hydrogen, alkyl,        or aryl and R²⁷ is alkyl or aryl;    -   X is absent or is —O—, —NR²⁸—, or —S—; and    -   n and m are independently 0, 1, 2, 3, 4, 5, or 6;    -   with the proviso that if R⁴ is halogen, R⁵ is hydrogen, and R¹        and R² are independently hydrogen, methoxy, saturated alkyl,        3-carboxy-4-chlorophenylamino, —N(CH₂CH₂OH)₂, or OC(O)Ph, then        R³ is not hydrogen, saturated alkyl, methoxy, halogen,        carboxy-4-chlorophenylamino, —N(CH₂CH₂OH)₂, or OC(O)Ph.

In other embodiments, the invention is directed towards compoundsrepresented by formula (I), (II), or (III):

-   -   wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, and R¹²        are each independently hydrogen, alkyl, hydroxyl, alkoxy,        —NR¹³R¹⁴, halo, nitro, cyano, borono, aryl, aryloxy,        —(CH₂)_(n)COOR¹⁵, —O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷, —CR¹⁸═NOH,        —CR¹⁹R²⁰NHOH, —SO₃H, —SO₂R²¹, —SO₂NHR²², —O(CH₂)_(m)OR²³,        —C(OH)═N(OH), —C(O)NR²⁴OH, —CHR²⁵N(COR²⁶)OH, or —C(O)R²⁷ or R⁴        and R⁵ together form —NR²⁸C(O)—, —C(O)NR²⁹—, —C(O)O—, or        —S(O)₂NR³⁰—, or R⁸ and R⁹ together form —O—, —NHC(O)—, —C(O)NH—,        —C(O)O—, —NR²⁹—, or —S(O)₂NH—;    -   R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹R²², R²³, R²⁴, R²⁵,        R²⁶, R²⁸, R²⁹, and R³⁰ are each independently hydrogen, alkyl,        or aryl and R²⁷ is alkyl or aryl;    -   X is absent or is —O—, —NR²⁸—, or —S—; and    -   n and m are independently 0, 1, 2, 3, 4, 5, or 6;    -   with the proviso that if R⁴ is halogen, R⁵ is hydrogen, and R¹        and R² are independently hydrogen, methoxy, saturated alkyl,        3-carboxy-4-chlorophenylamino, —N(CH₂CH₂OH)₂, or OC(O)Ph, then        R³ is not hydrogen, saturated alkyl, methoxy, halogen,        carboxy-4-chlorophenylamino, —N(CH₂CH₂OH)₂, or OC(O)Ph.

In other embodiments, the invention is directed to a compoundrepresented by the formula:

-   -   wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, and R¹²        are each independently hydrogen, alkyl, hydroxyl, alkoxy,        —NR¹³R¹⁴, halo, nitro, cyano, borono, aryl, aryloxy,        —(CH₂)_(n)COOR¹⁵, —O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷, —CR¹⁸═NOH,        —CR¹⁹R²⁰NHOH, —SO₃H, —SO₂R²¹, —SO₂NHR²², —O(CH₂)_(m)OR¹³,        —C(OH)═N(OH), —C(O)NR²⁴OH, —CHR²⁵N(COR²⁶)OH, or —C(O)R²⁷ or R⁴        and R⁵ together form —NR²⁸C(O)—, —C(O)NR²⁹—, —C(O)O—, or        —S(O)₂NR³⁰—, or R⁸ and R⁹ together form —O—, —NHC(O)—, —C(O)NH—,        —C(O)O—, —NR²⁹—, or —S(O)₂NH—;    -   R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵,        R²⁶, R²⁸, R²⁹, and R³⁰ are each independently hydrogen, alkyl,        or aryl and R²⁷ is alkyl or aryl;    -   X is absent or is —O—, —NR²⁸—, or —S—; and    -   n and m are independently 0, 1, 2, 3, 4, 5, or 6;    -   with the proviso that if R⁴ is halogen, R⁵ is hydrogen, and R¹        and R² are independently hydrogen, methoxy, saturated alkyl,        3-carboxy-4-chlorophenylamino, —N(CH₂CH₂OH)₂, or OC(O)Ph, then        R³ is not hydrogen, saturated alkyl, methoxy, halogen,        carboxy-4-chlorophenylamino, —N(CH₂CH₂OH)₂, or OC(O)Ph;    -   with the proviso that the compound is not:

or a salt thereof.

In other embodiments, the invention is directed to a compoundrepresented by the structure:

-   -   wherein R¹ is —O(CH₂)_(n)COOR⁹ or —OC(O)CH₂R³¹;    -   R², R³, R⁴, and R⁵ are each independently hydrogen, alkyl,        hydroxyl, alkoxy, —NR¹³R¹⁴, halo, nitro, cyano, borono, aryl,        aryloxy, —(CH₂)_(n)COOR¹⁵, —O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷,        —CR¹⁸═NOH, —CR¹⁹R²⁰NHOH, —SO₃H, —SO₂R²¹, —SO₂NHR²²,        —O(CH₂)_(m)OR²³, —C(OH)═N(OH), —C(O)NR²⁴OH, —CHR²⁵N(COR¹⁶)OH, or        —C(O)R²⁷ or R⁴ and R⁵ together form —NR²⁸C(O)—, —C(O)NR²⁹—,        —C(O)O—, or —S(O)₂NR³¹—;    -   R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹R²², R²³, R²⁴, R²⁵,        R²⁶R²⁸, R²⁹, and R³⁰ are each independently hydrogen, alkyl, or        aryl and R²⁷ is alkyl or aryl;    -   R³¹ is halogen; and    -   n and m are independently 0, 1, 2, 3, 4, 5, or 6;    -   with the proviso that the compound is not:

or a salt thereof.

In other embodiments, the invention is directed to a compoundrepresented by the formula:

-   -   I-9 or a salt thereof.

In other embodiments, the invention is directed to a compoundrepresented by the formula:

-   -   wherein R⁶, R⁷, and R⁹ are each independently hydrogen, C₁₋₆        alkyl, C₃₋₇ cycloalkyl, hydroxyl, C₁₋₆ alkoxy, —NR¹³R¹⁴,        halogen, nitro, cyano, borono, phenyl, benzyl, benzoyl, phenoxy,        benzyloxy, —(CH₂)_(n)COOR¹⁵, —O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷,        —CH═NOH, —CH₂NHOH, —SO₃H, —SO₂CH₃, —SO₂NHR²², —O(CH₂)_(m)OR²³,        —C(OH)═N(OH), —C(O)NR²⁴OH, —CHR²⁵N(COR²⁶)OH, or —C(O)R²⁷ or R⁸        and R⁹ together form —NHC(O)—, —C(O)NH—, —C(O)O—, —O—, —NR²⁹—,        or —S(O)₂NH—;    -   R⁸ is NH₂;    -   X is absent or is —O—, —NR²⁸—, or —S—;    -   R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R²², R²³, R²⁴, R²⁵, R²⁶, R²⁸, and R²⁹        are each independently hydrogen, alkyl, aryl, or cycloalkyl and        R²⁷ is alkyl, aryl, or cycloalkyl; and    -   n and m are independently 0, 1, 2, 3, 4, 5, or 6.

In other embodiments, the invention is directed to a compoundrepresented by the formula:

-   -   wherein R¹⁰, R¹¹, and R¹² are each independently hydrogen,        alkyl, hydroxyl, alkoxy, —NR¹³R¹⁴, halo, nitro, cyano, borono,        aryl, aryloxy, —(CH₂)_(n)COOR¹⁵, —O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷,        —CR¹⁸═NOH, —CR¹⁹R²⁰NHOH, —SO₃H, —SO₂R²¹, —SO₂NHR²²,        —O(CH₂)_(m)OR²³, —C(OH)═N(OH), —C(O)NR²⁴OH, —CHR²⁵N(COR²⁶)OH, or        —C(O)R²⁷;    -   R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵,        and R²⁶ are each independently hydrogen, alkyl, or aryl and R²⁷        is alkyl or aryl; and    -   n and m are independently 0, 1, 2, 3, 4, 5, or 6.

In certain embodiments, the invention is directed to a compound that isa tumor necrosis factor receptor 1 (TNF-R1) inhibitor that binds to anallosteric site of TNF-R1 with an affinity of 1000 nM or greateraffinity, preferably 100 nM or greater affinity, and more preferably of10 nM or greater affinity.

In certain embodiments, the invention is directed to one of theaforementioned compounds, or a compound different from theaforementioned compounds, that exhibits an affinity for wild type TNF-R1that is at least about 10-fold greater than the affinity the compoundexhibits for TNF-R1 bearing a substitution of an amino acid selectedfrom the group consisting of K35, G36, C55, E56, S57, G58, S59, F60,T61, A62, S63, C70, L71, S72, C73, S74, K75, C76, R77, K78, E79, M80,G81, Q82, V83, E84, I85, V90, D91, R92, D93, T94, V95, C96, G97, C98,R99, K100, N101, Q102, Y103, R104, H105, Y106, S108, E109, N110, L111,F112, Q113, C114, F115, Q130, E131, K132, and Q133.

In certain embodiments, the invention is directed to a TNF-R1 inhibitorcompound that: (i) binds to an allosteric site of TNF-R1 with anaffinity of 100 nM or greater affinity; and (ii) reduces the TNF-αmediated activation of NF-κB and p38 kinase when administered to a cell,compared to the TNF-α mediated activation of NF-κB and p38 kinaseactivity obtained in said cell when said compound is not administered tosaid cell.

In other embodiments, the invention is directed to compounds asdescribed above, with the proviso the compound is not a compound ofFormula I

-   -   wherein    -   R¹ and R² are independently hydrogen, methoxy, saturated alkyl,        3-carboxy-4-chlorophenylamino, —N(CH₂CH₂OH)₂, or OC(O)Ph,    -   R³ is hydrogen, ethyl, methoxy, halogen, t-butyl,        carboxy-4-chlorophenylamino, —N(CH₂CH₂OH)₂, or OC(O)Ph,    -   R⁴ is halogen, and    -   R⁵ is hydrogen.

In other embodiments, the invention is directed to a compoundrepresented by a formula (I), (II), or (III), or a pharmaceuticallyacceptable salt thereof:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, and R¹² are eachindependently alkyl, hydroxyl, alkoxy, —NR¹³R¹⁴, halo, nitro, cyano,borono, aryl, aryloxy, —(CH₂)_(n)COOR¹⁵, —O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷,—CR¹⁸═NOH, —CR¹⁹R²⁰NHOH, —SO₃H, —SO₂R²¹, —SO₂NHR²², —O(CH₂)_(m)OR²³,—C(OH)═N(OH), —C(O)NR²⁴OH, —CHR²⁵N(COR²⁶)OH, or —C(O)R²⁷ or R⁴ and R⁵together form —NR²⁸C(O)—, —C(O)NR²⁹—, —C(O)O—, or —S(O)₂NR³⁰—, or R⁸ andR⁹ together form —O—, —NHC(O)—, —C(O)NH—, —C(O)O—, —NR²⁹—, or —S(O)₂NH—;or R⁸ and R⁹ are each independently hydrogen;

R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵, R²⁶,R²⁸, R²⁹, and R³⁰ are each independently hydrogen, alkyl, or aryl andR²⁷ is alkyl or aryl;

X is absent or is —O—, —NR²⁸—, or —S—; and

n and m are independently 0, 1, 2, 3, 4, 5, or 6;

q, r, s, t, u, v, w, and x, are independently 0, 1, 2, or 3;

with a first proviso that if R⁴ is halogen, R⁵ is hydrogen, and R¹ andR² are independently hydrogen, methoxy, saturated alkyl,3-carboxy-4-chlorophenylamino, —N(CH₂CH₂OH)₂, or OC(O)Ph, then R³ is nothydrogen, saturated alkyl, methoxy, halogen,carboxy-4-chlorophenylamino, —N(CH₂CH₂OH)₂, or OC(O)Ph;

and with a second proviso that the compound is not:

-   -   I-9 or a salt thereof.

In yet other embodiments, the invention is directed to a compoundrepresented by the formula, or a pharmaceutically acceptable saltthereof:

wherein R¹ is —O(CH₂)_(n)COOR⁹ or —OC(O)CH₂R³¹;

R², R³, R⁴, and R⁵ are each independently alkyl, hydroxyl, alkoxy,—NR¹³R¹⁴, halo, nitro, cyano, borono, aryl, aryloxy, —(CH₂)_(n)COOR¹⁵,—O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷, —CR¹⁸═NOH, —CR¹⁹R²⁰NHOH, —SO₃H, —SO₂R²¹,—SO₂NHR²², —O(CH₂)_(m)OR²³, —C(OH)═N(OH), —C(O)NR²⁴OH, —CHR²⁵N(COR²⁶)OH,or —C(O)R²⁷ or R⁴ and R⁵ together form —NR²⁸C(O—, —C(O)NR²⁹—, —C(O)O—,or —S(O)₂NR³⁰—;

R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵, R²⁶,R²⁸, R²⁹, and R³⁰ are each independently hydrogen, alkyl, or aryl andR²⁷ is alkyl or aryl;

R³¹ is halogen; and

n and m are independently 0, 1, 2, 3, 4, 5, or 6;

q, r, and s are each independently 0, 1, 2 or 3

with the proviso that the compound is not:

or a salt thereof.

In yet other embodiments, the invention is directed to a compoundrepresented by the formula, or a pharmaceutically acceptable saltthereof:

wherein R⁶, R⁷, and R⁹ are each independently C₁₋₆ alkyl, C₃₋₇cycloalkyl, hydroxyl, C₁₋₆ alkoxy, —NR¹³R¹⁴, halogen, nitro, cyano,borono, phenyl, benzyl, benzoyl, phenoxy, benzyloxy, —(CH₂)_(n)COOR¹⁵,—O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷, —CH—NOH, —CH₂NHOH, —SO₃H, —SO₂CH₃,—SO₂NHR²², —O(CH₂)_(m)OR²³, —C(OH)═N(OH), —C(O)NR²⁴OH, —CHR²⁵N(COR²⁶)OH,or —C(O)R²⁷ or R⁸ and R⁹ together form-NHC(O)—, —C(O)NH—, —C(O)O—, —O—,—NR²⁹—, or —S(O)₂NH—;

or R⁸ is NH₂ or R⁸ and R⁹ are each independently hydrogen;

X is absent or is —O—, —NR²⁸—, or —S—;

R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R²², R²³, R²⁴, R²⁵, R²⁶, R²⁸, and R²⁹ are eachindependently hydrogen, alkyl, aryl, or cycloalkyl and R²⁷ is alkyl,aryl, or cycloalkyl;

t and u are each independently 0, 1, 2, or 3; and

n and m are independently 0, 1, 2, 3, 4, 5, or 6.

In yet other embodiments, the invention is directed to a compoundrepresented by the formula, or a pharmaceutically acceptable saltthereof:

wherein R¹⁰, R¹¹, and R¹² are each independently alkyl, hydroxyl,alkoxy, —NR¹³R¹⁴, halo, nitro, cyano, borono, aryl, aryloxy,—(CH₂)_(n)COOR¹⁵, —O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷, —CR¹⁸═NOH, —CR¹⁹R²⁰NHOH,—SO₃H, —SO₂R²¹, —SO₂NHR²², —O(CH₂)_(m)OR²³, —C(OH)═N(OH), —C(O)NR²⁴OH,—CHR²⁵N(COR²⁶)OH, or —C(O)R²⁷;

R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁵ and R²⁶ eachindependently hydrogen, alkyl, or aryl and R²⁷ is alkyl or aryl;

v, w, and x, are each independently 0, 1, 2, or 3 and

n and m are independently 0, 1, 2, 3, 4, 5, or 6.

In other embodiments, the invention is directed to a pharmaceuticalcomposition comprising a therapeutically effective amount of any of theaforementioned compounds and a pharmaceutically acceptable excipient.

In other embodiments, the invention is directed to methods of treatmentof a TNF-α mediated condition, comprising administering an effectiveamount any of the aforementioned compounds or compositions to a patientin need of such treatment. In other embodiments, the invention isdirected to methods of inhibiting tumor necrosis factor action,comprising administering an effective amount of any of theaforementioned compounds or compositions to a patient in need of suchtreatment. Preferred embodiments of the invention include methods oftreating arthritis, inflammation, psoriasis, or an autoimmune conditioncomprising administering an effective amount of any of theaforementioned compounds or compositions to a patient in need of suchtreatment.

In yet other embodiments, the invention is directed to use of any of theaforementioned compounds or compositions in the manufacture of amedicament for the therapeutic and/or prophylactic treatment of anautoimmune condition, including conditions such as arthritis,inflammation, and psoriasis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of isothermal titration calorimetry measurementsillustrating the binding of TNF-R1 inhibitor I-9 to (A) recombinant wildtype human TNF-R1 receptor and (B) mutant human TNF-R1.

FIG. 2 shows a Stern-Volmer plot for quenching of the intrinsictryptophan fluorescence of TNF-R1 by acrylamide for TNF-R1 alone (solidcircles) and in the presence of TNF-R1 inhibitor I-9 (open triangles).

FIG. 3 shows the results of experiments designed to show the effect ofTNF-R1 inhibitor I-9 on cell signaling. (A) Effect of I-9 onTNFα-induced phosphorylation of P38 in L929 cells; (B) Effect of I-9 onEGF-induced MAPK activation in NE91 cells; and (C) Effect of I-9 onsignaling in THP1. Cells were treated with vehicle (lane 1), LPS (10ng/mL, lane 2), TNF-α (100 ng/mL, lane 3), inhibitor I-9 (20 μg/mL, lane4), LPS (10 ng/mL)+inhibitor I-9 (20 μg/mL) (lane 5), and TNF-α (100ng/mL)+inhibitor I-9 (20 μg/mL) (lane 6).

FIG. 4 shows the results of an experiment designed to show the effect ofTNF-R1 inhibitor I-9 on collagen induced arthritis in a mouse model ofhuman rheumatoid arthritis. Data are expressed as mean±SEM.Probabilities are results of student t test, from comparisons withcontrol group mice treated with vehicle.

DETAILED DESCRIPTION

The present inventions are directed to the area of compounds and methodsfor inhibiting functions mediated by tumor necrosis factor. Suchcompounds and methods can also be used in treating diseases, disorders,and conditions in which tumor necrosis factor is a participant.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization described below are those well known andcommonly employed in the art. Standard techniques are used for nucleicacid and peptide synthesis. Generally, enzymatic reactions andpurification steps are performed according to the manufacturer'sspecifications. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences that are provided throughout this document. The nomenclatureused herein and the laboratory procedures in analytical chemistry, andorganic synthetic chemistry described below are those well known andcommonly employed in the art. Standard techniques, or modificationsthereof, are used for chemical syntheses and chemical analyses.

One embodiment of a suitable compound for a pharmaceutical compositionis represented by formula (I), or is a pharmaceutically acceptable saltthereof:

R¹ through R⁵ can be selected in view of factors such as, for example,affinity, activity, absorption, distribution, metabolism, excretion,pharmacokinetic, toxicological and other properties conducive to theiruse as pharmaceuticals.

In another embodiment, the compound is represented by formula (I), or isa pharmaceutically acceptable salt thereof, where R¹, R², R³, R⁴, and R⁵are each independently hydrogen, alkyl (preferably, saturated alkyl),hydroxyl, alkoxy, —NR¹³R¹⁴, halo, nitro, cyano, borono, aryl, aryloxy,—(CH₂)_(n)COOR¹⁵, —O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷, —CR¹⁸═NOH, —CR¹⁹R²⁰NHOH,—SO₃H, —SO₂R²¹, —SO₂NHR²², —O(CH₂)_(m)OR²³, —C(OH)═N(OH), —C(O)NR²⁴OH,—CHR²⁵N(COR²⁶)OH, or —C(O)R²⁷ or R⁴ and R⁵ together form —NR²⁸C(O)—,—C(O)NR²⁹—, —C(O)O—, or —S(O)₂NR³⁰—;

-   -   R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵,        R²⁶, R²⁸, R²⁹, and R³⁰ are each independently hydrogen, alkyl        (preferably, saturated alkyl), or aryl and R²⁷ is alkyl        (preferably, saturated alkyl) or aryl; and    -   n and m are independently 0, 1, 2, 3, 4, 5, or 6,    -   with the proviso that if R⁴ is halogen, R⁵ is hydrogen, and R¹        and R² are independently hydrogen, methoxy, saturated alkyl,        3-carboxy-4-chlorophenylamino, —N(CH₂CH₂OH)₂, or OC(O)Ph, then        R³ is not hydrogen, saturated alkyl, methoxy, halo,        carboxy-4-chlorophenylamino, —N(CH₂CH₂OH)₂, or —OC(O)Ph.

Examples of such compounds include the following:

In another embodiment, the compound is represented by formula (I), or isa pharmaceutically acceptable salt thereof, where R¹, R², R³, R⁴, and R⁵are each independently hydrogen, alkyl (preferably, saturated alkyl),hydroxyl, alkoxy, —NR¹³R¹⁴, nitro, cyano, borono, aryl, aryloxy,—(CH₂)_(n)COOR¹⁵, —O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷, —CR¹⁸═NOH, —CR¹⁹R²⁰NHOH,—SO₃H, —SO₂R²¹, —SO₂NHR²², —O(CH₂)_(m)OR²³, —C(OH)═N(OH), —C(O)NR²⁴OH,—CHR²⁵N(COR²⁶)OH, or —C(O)R²⁷ or R⁴ and R⁵ together form —NR²⁸C(O)—,—C(O)NR²⁹—, —C(O)O—, or —S(O)₂NR³⁰—;

-   -   R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵,        R²⁶, R²⁸, R²⁹, and R³⁰ are each independently hydrogen, alkyl        (preferably, saturated alkyl), or aryl and R²⁷ is alkyl        preferably, saturated alkyl) or aryl; and    -   n and m are independently 0, 1, 2, 3, 4, 5, or 6. Preferably, n        and m are 0, 1, or 2.

In yet another embodiment, the compound is represented by formula (I),or is a pharmaceutically acceptable salt thereof, where R¹, R², R³, R⁴,and R⁵ are each independently hydrogen, C₁₋₆ saturated alkyl, C₃₋₇cycloalkyl, hydroxyl, C₁₋₆ alkoxy, —NR¹³R¹⁴, nitro, cyano, borono,phenyl, benzyl, benzoyl, phenoxy, benzyloxy, —(CH₂)_(n)COOR¹⁵,—O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷, —CH═NOH, —CH₂NHOH, —SO₃H, —SO₂CH₃,—SO₂NHR²², —O(CH₂)_(m)OR²³, —C(OH)—N(OH), —C(O)NR²⁴OH, —CHR²⁵N(COR²⁶)OH,or —C(O)R²⁷ or R⁴ and R⁵ together form —NR²⁸C(O)—, —C(O)NR²⁹—, —C(O)O—,or —S(O)₂NR³⁰—;

-   -   R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴R²⁵,        R²⁶, R²⁸, R²⁹, and R³⁰ are each independently hydrogen, alkyl        (preferably, saturated alkyl), or aryl and R²⁷ is alkyl        (preferably, saturated alkyl) or aryl; and    -   n and m are independently 0, 1, 2, 3, 4, 5, or 6. Preferably, n        and m are 0, 1, or 2.

In another embodiment, the compound is represented by formula (I), or isa pharmaceutically acceptable salt thereof, where R¹, R², R³, R⁴, and R⁵are each independently hydrogen, methyl, ethyl, methoxy, —OC(O)H,—OC(O)CH₃, —OC(O)CH₂Cl, hydroxyl, —NH₂, —N(CH₃)₂, —OC(O)CHCH₂, or—OCH₂COOCH₃ or R⁴ and R⁵ together form —NHC(O)—.

In yet another embodiment, the compound is represented by formula (I),or is a pharmaceutically acceptable salt thereof, where R¹, R², and R³are each independently hydrogen, methoxy, —OC(O)H, —OC(O)CH₃,—OC(O)CH₂Cl, hydroxyl, —NH₂, —N(CH₃)₂, —OC(O)CHCH₂, —OCH₂COOCH₃; R⁴ ishydrogen; and R⁵ is hydrogen, methyl, —OC(O)H, or hydroxyl or thecompound is a pharmaceutically acceptable salt thereof.

In yet another embodiment, the compound is represented by (I), or is apharmaceutically acceptable salt thereof, where R¹ is —O(CH₂)_(n)COOR⁹(for example, —OCH₂COOCH₃) or —OC(O)CH₂C1 and R², R³, R⁴, R⁵ and R⁹ areas defined in the first embodiment of formula (I) above, with theproviso that R¹, R², and R³ are not all —OCH₂COOCH₃. Examples of suchcompounds include compounds I-11, I-12, I-13, I-14, and I-15 above.

In another embodiment, the compound is represented by formula (I), or isa pharmaceutically acceptable salt thereof, where R¹ is —OCH₂COOCH₃ or—OC(O)CH₂Cl, R² is —OCH₂COOCH₃, hydroxyl, or —OC(O)CH₂Cl; and R³ ishydrogen, —OCH₂COOCH₃, hydroxyl, —OC(O)CH₃, or —OC(O)CH₂Cl, with theproviso that R¹, R², and R³ are not all —OCH₂COOCH₃. Preferably, R⁴ ishydrogen and R⁵ is hydrogen, methyl, hydroxyl, or —COOH. Examples ofsuch compounds include compounds having formulas I-11 to I-15.

Other suitable compounds for the pharmaceutical composition includecompounds represented by formula (II), or pharmaceutically acceptablesalts thereof:

R⁶ through R⁹ can be selected in view of factors such as, for example,affinity, activity, absorption, distribution, metabolism, excretion,pharmacokinetic, toxicological and other properties conducive to theiruse as pharmaceuticals.

In one embodiment, the compound is represented by formula (II), or is apharmaceutically acceptable salt thereof, where X is absent or is —O—,—NR²⁸—, or —S—; R⁶, R⁷, R⁸, and R⁹ are each independently hydrogen,alkyl (preferably, saturated alkyl), hydroxyl, alkoxy, —NR¹³R¹⁴, halo,nitro, cyano, borono, aryl, aryloxy, —(CH₂)_(n)COOR¹⁵,—O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷, —CR¹⁸═NOH, —CR¹⁹R²⁰NHOH, —SO₃H, —SO₂R²¹,SO₂NHR²², —O(CH₂)_(m)OR²³, —C(OH)═N(OH), —C(O)NR²⁴OH, —CHR²⁵N(COR²⁶)OH,or —C(O)R²⁷ or R⁸ and R⁹ together form —NHC(O)—, —C(O)NH—, —C(O)O—, —O—,—NR²⁹—, or —S(O)₂NH—;

-   -   R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵,        R²⁶, R²⁸, and R²⁹ are each independently hydrogen, alkyl        (preferably, saturated alkyl), or aryl and R²⁷ is alkyl        (preferably, saturated alkyl) or aryl; and    -   n and m are independently 0, 1, 2, 3, 4, 5, or 6.

Examples of such compounds include:

In another embodiment, the compound is represented by formula (II), oris a pharmaceutically acceptable salt thereof, where X is absent or is—O—, —NR²⁸—, or —S—; R⁶, R⁷, R⁸, and R⁹ are each independently hydrogen,C₁₋₆ saturated alkyl, C₃₋₇ cycloalkyl, hydroxyl, C₁₋₆ alkoxy, —NR¹³R¹⁴,halogen, nitro, cyano, borono, phenyl, benzyl, benzoyl, phenoxy,benzyloxy, —(CH₂)_(n)COOR¹⁵, —O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷, —CH═NOH,—CH₂NHOH, —SO₃H, —SO₂CH₃, —SO₂NHR²², —O(CH₂)_(m)OR²³, —C(OH)═N(OH),—C(O)NR²⁴OH, —CHR²⁵N(COR²⁶)OH, or —C(O)R²⁷ or R⁸ and R⁹ together form—NHC(O)—, —C(O)NH—, —C(O)O—, —O—, —NR²⁹—, or —S(O)₂NH—;

-   -   R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R²², R²³, R²⁴, R²⁵, R²⁶, R²⁸, and R²⁹        are each independently hydrogen, alkyl (preferably, saturated        alkyl), or aryl and R²⁷ is alkyl (preferably, saturated alkyl)        or aryl; and    -   n and m are independently 0, 1, 2, 3, 4, 5, or 6. Preferably, n        and m are 0, 1, or 2.

In another embodiment, the compound is represented by formula (II), oris a pharmaceutically acceptable salt thereof, where X is absent or is—O—, —NR²⁸—, or —S—, R⁶, R⁷, R⁸, and R⁹ are each independently hydrogen,—C(O)OH, —C(O)OCH₃, hydroxyl, —NH₂, or halo, or R⁸ and R⁹ together form—O—.

In yet another embodiment, the compound is represented by formula (II),or is a pharmaceutically acceptable salt thereof, where X is absent oris —O—, R⁸ is —NH₂ and R⁶, R⁷, and R⁹ are as described above for thefirst embodiment of compounds of formula (II). Preferably, X is —O—, R⁶and R⁷ are halo, and R⁹ is hydrogen. An example of such a compound iscompound II-6.

In another embodiment, the compound is a pharmaceutically acceptablemonovalent salt of a compound having the structure of formula (II) whereX is absent or is —O—, R⁸ is —C(O)OR¹⁵, R⁶ and R⁷ are hydroxyl, and R⁹is hydrogen.

Other suitable compounds for use in the pharmaceutical compositionsinclude compounds represented by formula (III), or pharmaceuticallyacceptable salts thereof:

-   -   or pharmaceutically acceptable salts thereof. R¹⁰ through R¹²        can be selected in view of factors such as, for example,        affinity, activity, absorption, distribution, metabolism,        excretion, pharmacokinetic, toxicological and other properties        conducive to their use as pharmaceuticals.

In one embodiment, the compound is represented by formula (III), or is apharmaceutically acceptable salt thereof, where R¹⁰, R¹¹, and R¹² areeach independently hydrogen, alkyl (preferably, saturated alkyl),alkoxy, —NR¹³R¹⁴, halo, nitro, cyano, borono, aryl, aryloxy,—(CH₂)_(n)COOR¹⁵, —O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷, —CR¹⁸═NOH, —CR¹⁹R²⁰NHOH,—SO₃H, —SO₂R²¹, —SO₂NHR²², —O(CH₂)_(m)OR²³, —C(OH)═N(OH), —C(O)NR²⁴OH,—CHR²⁵N(COR²⁶)OH, or —C(O)R²⁷;

-   -   R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵,        and R²⁶ are each independently hydrogen, alkyl (preferably,        saturated alkyl), or aryl and R²⁷ is alkyl (preferably,        saturated alkyl) or aryl; and    -   n and m are independently 0, 1, 2, 3, 4, 5, or 6.

Examples of such compounds include:

In another embodiment, the compound is represented by formula (III), oris a pharmaceutically acceptable salt thereof, where R¹⁰, R¹¹, and R¹²are each independently hydrogen, C₁₋₆ saturated alkyl, C₃₋₇ cycloalkyl,hydroxyl, C₁₋₆ alkoxy, —NR¹³R¹⁴, halogen, nitro, cyano, borono, phenyl,benzyl, benzoyl, phenoxy, benzyloxy, —(CH₂)_(n)COOR¹⁵,—O(CH₂)_(n)COOR¹⁶, —OC(O)R¹⁷, —CH—NOH, —CH₂NHOH, —SO₃H, —SO₂CH₃,—SO₂NHR²², —O(CH₂)_(m)OR²³, —C(OH)═N(OH), —C(O)NR²⁴OH, —CHR²⁵N(COR²⁶)OH,or —C(O)R²⁷;

-   -   R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R²², R²³, R²⁴, R²⁵, and R²⁶ are each        independently hydrogen, alkyl (preferably, saturated alkyl), or        aryl and R²⁷ is alkyl (preferably, saturated alkyl) or aryl; and    -   n and m are independently 0, 1, 2, 3, 4, 5, or 6. Preferably, n        and m are 0, 1, or 2.

In another embodiment, the compound is represented by formula (III), oris a pharmaceutically acceptable salt thereof, where R¹⁰, R¹¹, and R¹²are each independently hydroxyl, —OC(O)H, —OC(O)CH₃, halo, or nitro.

In yet another embodiment, the compound is represented by formula (III),or is a pharmaceutically acceptable salt thereof, where R¹² is nitro andR¹⁰ and R¹¹ are hydroxyl, —OC(O)CH₃, or halo.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon group (cycloalkyl), or combination thereof, which may befully saturated, mono- or polyunsaturated and can include di- andmultivalent radicals, and can have a number of carbon atoms optionallydesignated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon groups include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotonyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups, which are limited to hydrocarbon groups aretermed “homoalkyl”. Alkyl groups include, for example, C₁₋₆unsubstituted alkyl, C₃₋₇ unsubstituted cycloalkyl, trifluoromethyl,chloromethyl, and hydroxymethyl.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon group, or combinations thereof, consisting of anumber of carbon atoms and at least one heteroatom selected from thegroup consisting of O, N, Si and S, and wherein the nitrogen, carbon andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.

The term “alkoxy” is used in its conventional sense, and refers to thosealkyl groups attached to the remainder of the molecule via an oxygenatom. Alkoxy groups include, but are not limited to, trifluoromethoxyand difluoromethoxy.

The term “cycloalkyl”, by itself or in combination with other terms,represents, unless otherwise stated, cyclic versions of substituted orunsubstituted “alkyl” and substituted or unsubstituted “heteroalkyl”(“heterocycloalkyl”). For heterocycloalkyl, a heteroatom can occupy theposition at which the heterocycle is attached to the remainder of themolecule. Examples of cycloalkyl include, but are not limited to,cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl,and the like. Examples of heterocycloalkyl include, but are not limitedto, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. The heteroatoms and carbonatoms of the cyclic structures are optionally oxidized or, in the caseof N, quaternized.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom.

The term “aryl” means, unless otherwise stated, a substituted orunsubstituted polyunsaturated, aromatic, hydrocarbon substituent whichcan be a single ring or multiple rings (preferably from 1 to 3 rings)which are fused together or linked covalently. The term “heteroaryl”refers to aryl groups (or rings) that contain from one to fourheteroatoms selected from N, O, and S, wherein the nitrogen, carbon andsulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized. A heteroaryl group can be attached to theremainder of the molecule through a heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. “Aryl” and “heteroaryl” alsoencompass ring systems in which one or more non-aromatic ring systemsare fused, or otherwise bound, to an aryl or heteroaryl system.Aryl-containing groups include, but are not limited to, phenyl, phenoxy,phenoxycarbonyl, benzoyl, benzyl, and benzyloxy.

The term “aryloxy” is used in its conventional sense, and refers tothose aryl groups attached to the remainder of the molecule via anoxygen atom.

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) include both substituted and unsubstituted forms of theindicated group, unless indicated otherwise. Preferred substituents foreach type of group are provided below.

Substituents for the alkyl groups (including those groups often referredto as heteroalkyl, alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generally referred to as “alkyl substituents”and they can be one or more of a variety of groups selected from, butnot limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2 m′+1), where m′ is the totalnumber of carbon atoms in such group. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedalkyl including substituted or unsubstituted heteroalkyl, andsubstituted or unsubstituted aryl, e.g., aryl substituted with 1-3halogens. When a compound of the invention includes more than one Rgroup, for example, each of the R groups is independently selected asare each of the R′, R″, R′″ and R″″ groups when more than one of thesegroups is present.

Similar to the substituents described for alkyl groups, the arylsubstituents are generally referred to as “aryl substituents” and arevaried and selected from, for example: halogen, —OR′, ═O, ═NR′, —N—OR′,—NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN, —NO₂,—R′, and —N₃, in a number ranging from zero to the total number of openvalences on the aromatic ring system; and where R′, R″, R′″ and R″″ arepreferably independently selected from hydrogen, substituted orunsubstituted alkyl including substituted or unsubstituted heteroalkyl,and unsubstituted aryl. When a compound of the invention includes morethan one R group, for example, each of the R groups is independentlyselected as are each of the R′, R″, R′″ and R″″ groups when more thanone of these groups is present.

Aryl-containing groups include, but are not limited to, phenyl, phenoxy,phenoxycarbonyl, benzoyl, benzyl, and benzyloxy.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S), boron (B) and silicon (Si).

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

Tumor Necrosis Factor Receptor

Tumor necrosis factor (TNF) receptor is one of the central mediators ofinflammation. The three dimensional structure of the TNF receptor 1(TNF-R1) complex has been determined with and without its ligand. Smallmolecules, described below, can bind to a discrete surface cavity andcan disable ligand-induced TNF receptor functions. Although not wishingto be bound by any particular theory, it is thought that this is aconsequence of the conformational perturbation of a loop on the receptorcontaining tryptophan-107 (W107). The conformational perturbationapproach identifies surface sites that are relevant for TNF-α,receptor's biological activity in vitro and in vivo.

TNF-R1 is a transmembrane receptor glycoprotein of Mr approximately 55kDa. The primary translation product of TNF-R1 is modified by cleavageof an amino terminal signal sequence and further by cleavage betweenarginine and aspartic acid residues found, respectively, approximately11 and 12 amino acids from the signal sequence cleavage site. A solublefragment of TNF-R1 of approximately 20 kDa can be isolated from sera andurine. The soluble fragment retains TNF-R1 binding activity. As usedherein, the position of amino acids in TNF-R1 are given with referenceto the sequence shown in SEQ ID NO: 1.

Although not wishing to be bound by any particular theory, the crystalstructure analysis of the TNF receptor complex with and without ligandsdid not reveal any changes consistent with ligand induced fit. (Banneret al., Cell 73, 431 (1993)). Hence the structural role of the ligandwas postulated to bring the receptor together and facilitate receptoractivation.

Three contact sites (WP5, WP8 and WP9) on TNF-R1 have been identified ascontributing to stable ligand complex formation. WP9 (amino acids105-113) appears to be important for functional interaction with TNF-α.(Takasaki et al., Nature Biotechnology 15, 1266 (1997)). A flexiblehinge (G81 and G97) identified from the crystal structure analysis waspostulated to provide ligand induced conformational changes. Contrary tothe result predicted if such a flexible hinge existed, however, nosignificant conformational changes were observed in the crystallographiccomplex of TNF-R1/TNF-α versus TNF-R1 alone. Thus, the crystal studiesfailed to suggest the presence of an allosteric site or cavity onTNF-R1.

Small molecule ligands are identified herein that can be used to induceconformational perturbation at WP9. Identified molecules can then befurther selected and modified, if desired, based on their ability toinduce conformational changes using molecular simulation studies.Cavities and clefts on the surface of proteins distal to regulatorysites such as ligand binding sites or catalytic sites may be utilized tomodulate the function of proteins/receptors by inducing conformationalchanges as a consequence of lodging small molecules into the cavities.The mode of inhibition can share some features with that of allostericinhibitors and such small molecules can be referred to as “cavityinduced allosteric” inhibitors and the method can be termed as “cavityinduced allosteric modification” (CIAM). The target allosteric cavity inTNF-R1 is distal to the W9 contact site and is bounded by a concavesurface that can accommodate compounds. The allosteric cavity is boundedby amino acids K35-G36, C55 through S63, C70 through 185, V90 throughY106, S108 through F115 and Q130 through Q133 (see SEQ ID NO: 1).

Treatment of TNF Mediated Conditions

The term “therapeutic agent” is intended to mean a compound that, whenpresent in a therapeutically effective amount, produces a desiredtherapeutic effect on a mammal. For example, an “effective amount” of acompound for inhibiting tumor necrosis factor action is an amount of acompound or composition that is sufficient to inhibit, reduce, orotherwise mitigate an undesirable effect of tumor necrosis factoraction. Such inhibition may occur for example, and without limitation,via a direct interaction, and/or through a competitive interaction, orvia an allosteric interaction with TNF-R1, TNF-α, or with anotherbinding protein.

Pharmaceutical compositions containing the small molecules describedbelow can be useful to treat individuals suffering from TNF-mediateddiseases, disorders, and conditions. Examples of TNF-mediated diseases,disorders, and conditions include inflammatory diseases and autoimmunediseases such as rheumatoid arthritis (RA), multiple sclerosis (MS),Sjogren's syndrome, sarcoidosis, insulin dependent diabetes mellitus(IDDM), autoimmune thyroiditis, reactive arthritis, ankylosingspondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis,vasculitis, Wegener's granulomatosis, Crohn's disease, ulcerativecolitis, Lupus (SLE), Grave's disease, myasthenia gravis, autoimmunehemolytic anemia, autoimmune thrombocytopenia, asthma, cryoglobulinemia,primary biliary sclerosis, pernicious anemia, and periodontal disease(e.g., gingivitis). Individuals suffering from such diseases, disorders,and conditions may be treated by administering to them a therapeuticallyeffective amount of a pharmaceutical composition that contains acompound having Formula I, II, or III or pharmaceutically acceptablesalt thereof. Examples of other compounds can be found in U.S. patentapplication Ser. No. 09/720,647, incorporated herein by reference.

Binding Properties of Compounds

Compounds of the invention bind to TNF-R1. Preferably, compounds bind toTNF-R1 with an affinity (e.g., Id) of 1 μM or less. Without limiting thepresent disclosure, binding activity may be determined by binding ofcompounds to cells that express TNF-R1 on their cell surface or abinding of compounds to purified or partially purified TNF-R1. Bindingmay be determined using, as non-limiting examples, native or recombinantTNF-R1, or fragments thereof. Binding of compounds may be determinedusing methods that are well known to those skilled in the art. Apreferred method for determining binding activity of compounds to TNF-R1is isothermal titration calorimetry.

In preferred embodiments, a compound exhibits at least about 10-foldgreater binding to wild type TNF-R1 or fragment thereof than the bindingthe compound exhibits for a mutant of TNF-R1 or mutant fragment thereof.More preferred are compounds that exhibit about 100-fold greater bindingto TNF-R1 or fragment thereof, compared to the binding the compoundexhibits for a mutant of TNF-R1 or mutant fragment thereof. Mostpreferred are compounds that exhibit about 1000-fold greater binding toTNF-R1 or fragment thereof, compared to the binding the compoundexhibits for a mutant of TNF-R1 or mutant fragment thereof.

Further preferred are compounds exhibiting the aforementioned greaterbinding to wild type TNF-R1 or fragment thereof compared to acorresponding mutant TNF-R1 or fragment thereof, wherein said mutantbears a substitution in an amino acid selected from the group consistingof K35, G36, C55, E56, S57, G58, S59, F60, T61, A62, S63, C70, L71, S72,C73, S74, K75, C76, R77, K78, E79, M80, G81, Q82, V83, E84, I85, V90,D91, R92, D93, T94, V95, C96, G97, C98, R99, K100, N101, Q102, Y103,R104, H105, Y106, S108, E109, N110, L111, F112, Q113, C114, F115, Q130,E131, K132, and Q133. Further preferred are mutants bearing asubstitution at Q82 or F112. Most preferred are mutants bearing asubstitution at Q82 and F112.

Biological Activity

The activity a compound of the invention can be measured using in vivoor vitro biological assays that measure, for example and withoutlimitation, the ability of a compound to interfere with the biologicalactivity of TNF-α. One example of such an assay is measuring the abilityof a compound to inhibit TNF-α mediated cytolysis in L929 cells, orother suitable cells. Other examples of such assays include assaying theability of a compound to block or inhibit an event associated withintracellular signaling following treatment with TNF-α. Examples of suchsignaling events include the phosphorylation of NFκB and/or p38 in L929cells and NE91 cells.

In one embodiment, the biological activity of a compound is measured bythe ability of the compound to inhibit TNF-α mediated cytolysis of L929cells. Compounds of the present invention include compounds that show a50% inhibition of TNF-α mediated cytolysis of L929 cells at aconcentration of 100 μM or less. Other compounds include those that showa 50% inhibition of TNF-α mediated cytolysis of L929 cells at aconcentration of 25 μM or less. Still other compounds show a 50%inhibition of TNF-α mediated cytolysis of L929 cells at a concentrationof 10 μM or less. Still other compounds show a 50% inhibition of TNF-αmediated cytolysis of L929 cells at a concentration selected from thegroup consisting of 1 μM or less, 100 nM or less, 10 nM or less, and 1nM or less.

In another embodiment, the biological activity of a compound is measuredby the ability of the compound to inhibit TNF-α-mediated cytolysis inhuman cell lines such as TMP1, or other suitable human or monkey celllines.

Salts and Derivatives

Various pharmaceutically acceptable salts, ether derivatives, esterderivatives, acid derivatives, and aqueous solubility alteringderivatives of the active compound also are encompassed by the presentinvention. The present invention further includes all individualenantiomers, diastereomers, racemates, and other isomers of thecompound. The invention also includes all polymorphs and solvates, suchas hydrates and those formed with organic solvents, of this compound.Such isomers, polymorphs, and solvates may be prepared by methods knownin the art, such as by regiospecific and/or enantioselective synthesisand resolution, based on the disclosure provided herein.

Suitable salts of the compound include, but are not limited to, acidaddition salts, such as those made with hydrochloric, hydrobromic,hydroiodic, hydrofluoric, perchloric, sulfuric, nitric, phosphoric,acetic, propionic, glycolic, lactic pyruvic, malonic, succinic, maleic,fumaric, malic, tartaric, citric, benzoic, carbonic cinnamic, mandelic,methanesulfonic, ethanesulfonic, hydroxyethanesulfonic,benezenesulfonic, p-toluene sulfonic, cyclohexanesulfamic, salicyclic,p-aminosalicylic, 2-phenoxybenzoic, and 2-acetoxybenzoic acid; saltsmade with saccharin; alkali metal salts, such as lithium, sodium, andpotassium salts; alkaline earth metal salts, such as calcium andmagnesium salts; salts formed from Lewis acids, such as borontrifluoride; and salts formed with organic or inorganic ligands, such asquaternary ammonium salts (for example, tris(hydroxymethyl)aminomethanesalts).

Additional suitable salts include, but are not limited to, acetate,benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate,bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate,citrate, dihydrochloride, edetate, edisylate, estolate, esylate,fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate,hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate,malate, maleate, mandelate, mesylate, methylbromide, methylnitrate,methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammoniumsalt, oleate, pamoate (embonate), palmitate, pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate, sulfate,subacetate, succinate, tannate, tartrate, teoclate, tosylate,triethiodide and valerate salts of the compound of the presentinvention.

Prodrugs and active metabolites of compounds disclosed herein are alsowithin the scope of the invention.

A prodrug is a pharmacologically inactive compound that is convertedinto a pharmacologically active agent by a metabolic transformation orany other chemical or biological process (e.g., hydrolysis). Forexample, in vivo, a prodrug can be acted on by naturally occurringenzyme(s) resulting in liberation of the pharmacologically active agent.Conventional procedures for the selection and preparation of suitableprodrug derivatives are described, for example, in “Design of Prodrugs,”ed. H. Bundgaard, Elsevier, 1985.

An active metabolite is a compound that results from metabolism ofanother compound after administration of the latter to a subject.Metabolites can be identified by techniques well-known in the art.

Formulation and Administration

Suitable dosage forms include but are not limited to oral, rectal,sub-lingual, mucosal, nasal, ophthalmic, subcutaneous, intramuscular,intravenous, transdermal, spinal, intrathecal, intra-articular,intra-arterial, sub-arachinoid, bronchial, lymphatic, and intra-uterilleadministration, and other dosage forms for systemic delivery of activeingredients. In a preferred embodiment, the dosage form is suitable fororal administration.

To prepare such pharmaceutical dosage forms, one or more of theaforementioned compounds of formulae (I), (II), or (III), or apharmaceutically acceptable salt thereof, are intimately admixed with apharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier may take a wide variety of formsdepending on the form of preparation desired for administration.

For parenteral formulations, the carrier will usually comprise sterilewater, though other ingredients, for example, ingredients that aidsolubility or for preservation, may be included. Injectable solutionsmay also be prepared in which case appropriate stabilizing agents may beemployed.

In preparing the compositions in oral dosage form, any of the usualpharmaceutical media may be employed. Thus, for liquid oralpreparations, such as, for example, suspensions, elixirs and solutions,suitable carriers and additives include water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents and the like. For solidoral preparations such as, for example, powders, capsules, caplets, andtablets, suitable carriers and additives include starches, sugars,diluents, granulating agents, lubricants, binders, disintegrating agentsand the like. Due to their ease in administration, tablets and capsulesrepresent the most advantageous oral dosage unit form. If desired,tablets may be sugar coated or enteric coated by standard techniques.

In some applications, it may be advantageous to utilize the active agentin a “vectorized” form, such as by encapsulation of the active agent ina liposome, micelle, or other encapsulant medium, or by fixation of theactive agent, e.g., by covalent bonding, chelation, assembly, orassociative coordination, on a suitable biomolecule, such as thoseselected from proteins, lipoproteins, glycoproteins, andpolysaccharides.

Treatment methods of the present invention using formulations suitablefor oral administration may be presented as discrete units such ascapsules, cachets, tablets, or lozenges, each containing a predeterminedamount of the active ingredient as a powder or granules. Optionally, asuspension in an aqueous liquor or a non-aqueous liquid may be employed,such as a syrup, an elixir, an emulsion, or a draught.

A tablet may be made by compression or molding, or wet granulation,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing in a suitable machine, with the activecompound being in a free-flowing form such as a powder or granules whichoptionally is mixed with a binder, disintegrant, lubricant, inertdiluent, surface active agent, or discharging agent. Molded tabletscomprised of a mixture of the powdered active compound with a suitablecarrier may be made by molding in a suitable machine.

A syrup may be made by adding the active compound to a concentratedaqueous solution of a sugar, for example sucrose, to which may also beadded any accessory ingredient(s). Such accessory ingredient(s) mayinclude flavorings, suitable preservative, agents to retardcrystallization of the sugar, and agents to increase the solubility ofany other ingredient, such as a polyhydroxy alcohol, for exampleglycerol or sorbitol.

Formulations suitable for parenteral administration usually comprise asterile aqueous preparation of the active compound, which preferably isisotonic with the blood of the recipient (e.g., physiological salinesolution). Such formulations may include suspending agents andthickening agents and liposomes or other microparticulate systems whichare designed to target the compound to blood components or one or moreorgans. The formulations may be presented in unit-dose or multi-doseform.

Parenteral administration may comprise any suitable form of systemicdelivery or delivery directly to the CNS. Administration may for examplebe intravenous, intra-arterial, intrathecal, intramuscular,subcutaneous, intramuscular, intra-abdominal (e.g., intraperitoneal),etc., and may be effected by infusion pumps (external or implantable) orany other suitable means appropriate to the desired administrationmodality.

Nasal and other mucosal spray formulations (e.g., inhalable forms) cancomprise purified aqueous solutions of the active compounds withpreservative agents and isotonic agents. Such formulations arepreferably adjusted to a pH and isotonic state compatible with the nasalor other mucous membranes. Alternatively, they can be in the form offinely divided solid powders suspended in a gas carrier. Suchformulations may be delivered by any suitable means or method, e.g., bynebulizer, atomizer, metered dose inhaler, or the like.

Formulations for rectal administration may be presented as a suppositorywith a suitable carrier such as cocoa butter, hydrogenated fats, orhydrogenated fatty carboxylic acids.

Transdermal formulations may be prepared by incorporating the activeagent in a thixotropic or gelatinous carrier such as a cellulosicmedium, e.g., methyl cellulose or hydroxyethyl cellulose, with theresulting formulation then being packed in a transdermal device adaptedto be secured in dermal contact with the skin of a wearer.

In addition to the aforementioned ingredients, formulations of thisinvention may further include one or more accessory ingredient(s)selected from diluents, buffers, flavoring agents, binders,disintegrants, surface active agents, thickeners, lubricants,preservatives (including antioxidants), and the like. Suchpharmaceutical compositions can be prepared by methods and containcarriers which are well-known in the art. A generally recognizedcompendium of such methods and ingredients is Remington: The Science andPractice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. LippingcottWilliams and Wilkins: Philadelphia, Pa., 2000.

The formulation of the present invention can have immediate release,sustained release, delayed-onset release or any other release profileknown to one skilled in the art.

The subject receiving the pharmaceutical composition is preferably ananimal, including, but not limited, to an animal such a cow, horse,sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, andguinea pig, and is more preferably a mammal, and most preferably ahuman.

The amount of the active agent to be administered can typically rangefrom between about 0.01 to about 25 mg/kg/day, preferably from betweenabout 0.1 to about 10 mg/kg/day and most preferably from between about0.2 to about 5 mg/kg/day. It will be understood that the pharmaceuticalformulations of the present invention need not necessarily contain theentire amount of the agent that is effective in treating the disorder,as such effective amounts can be reached by administration of aplurality of doses of such pharmaceutical formulations.

In a preferred embodiment of the present invention, the compounds areformulated in capsules or tablets, preferably containing 25 to 200 mg ofthe compounds of the invention, and are preferably administered to apatient at a total daily dose of about 0.5 mg to about 2 g, preferablyabout 7.5 mg to about 750 mg, more preferably about 15 mg to 750 mg, andmost preferably from about 50 to about 200 mg.

A pharmaceutical composition for parenteral administration contains fromabout 0.01% to about 100% by weight of the active agents of the presentinvention, based upon 100% weight of total pharmaceutical composition.

EXAMPLES

The following examples illustrate the invention, but are not limiting.

Synthesis of Select Compounds

1,1,1-Tris-(4-carboxymethoxyphenyl)ethane, trisodium salt of I-3

-   -   Aqueous sodium hydroxide (3 mL of 0.1 N, 0.3 mmol) was added to        a solution of 1,1,1-tris-(4-carboxymethoxyphenyl)ethane (48 mg,        0.1 mmol, made according to Hayakawa, T., et al., Polymer J.,        2000, 32(9), 784) in 10 mL of ethanol and the mixture was        stirred for 2 h at room temperature. The mixture was evaporated        to dryness under reduced pressure to give 55 mg (100% yield) of        the desired compound as a white solid, mp>300° C. ¹H NMR (300        MHz, D₂O): δ 1.85 [s, 3H, —CH₃], 4.18 [s, 2H, —OCH₂—], 6.67 [d,        2H, J=7.7 Hz, Ar—H], 6.83 [d, 2H, J=7.7 Hz, Ar—H].

1,1-Bis-(4-hydroxyphenyl)-1-(4-methoxycarbonylmethoxyphenyl)ethane(I-11)

Methyl bromoacetate (0.48 mL, 5 mmol) was added dropwise to an acetonesolution (10 mL) including 1,1,1-tris(4-hydroxyphenyl)ethane (1.53 g, 5mmol) potassium carbonate (0.79 g, 5 mmol) and potassium iodide (16 mg,0.1 mmol). The addition was done at room temperature under argon. Themixture was refluxed for 24 h, and then it was cooled to roomtemperature and extracted with ethyl acetate. The combined organic layerwas washed with 3% aqueous NaHCO₃, a saturated brine solution, dried(Na₂SO₄) and then evaporated to dryness to give 1.61 g (85% yield) ofthe desired compound as a colorless semisolid. ¹H-NMR (300 MHz,d₆-DMSO): 1.96 [s, 3H, —CH₃], 3.67 [s, 3H, —OCH₃], 4.71 [s, 4H, —OCH₂—],6.57-6.64 [m, 4H, Ar—H], 6.72-6.81 [m, 6H, Ar—H], 6.85-6.92 [m, 2H,Ar—H].

1,1-Bis-(4-carboxymethoxy-phenyl)-1-(4-hydroxyphenyl)ethane, trisodiumsalt of I-12

In step (i), methyl bromoacetate (0.95 mL, 10 mmol) was added dropwiseto a solution of 1,1,1-tris(4-hydroxyphenyl)ethane (1.53 g, 5 mmol),potassium carbonate (1.38 g, 10 mmol) and potassium iodide (16 mg, 0.1mmol) in 10 mL of acetone at room temperature under argon. The mixturewas refluxed for an additional 24 h; after cooling to room temperaturethe solution was extracted with ethyl acetate. The combined organiclayer was washed with 3% aqueous NaHCO₃, saturated brine solution, dried(Na₂SO₄) and then evaporated to dryness to afford a solid, which wasused without further purification in step (ii).

In step (ii), 0.1 N aqueous sodium hydroxide (9 mL, 0.9 mmol) was addedto a solution of 132 mg of the product from step (i) in 5 mL of methanoland the mixture was stirred for an additional 2 h at room temperature.The precipitate was filtered, rinsed with cold ethanol and dried to give133 mg (91% yield) of the desired compound as a white solid, mp>300° C.¹H NMR (300 MHz, D₂O): δ 1.77 [s, 3H, —CH₃], 4.20 [s, 4H, —OCH₂—], 6.35[d, 1H, J=9.5 Hz, Ar—H], 6.54-6.66 [m, 6H, Ar—H], 6.75-6.86 [m, 5H,Ar—H].

1,1,1-Tris-(4-chloroacetoxyphenyl)ethane (I-13)

Chloroacetyl chloride (0.12 mL, 1.5 mmol) was added dropwise to an icecooled solution of 1,1,1-tris(4-hydroxyphenyl)ethane (0.15 g, 0.5 mmol)and triethylamine (0.22 mL, 1.5 mmol) in 10 mL of CH₂Cl₂. The mixturewas allowed to stand at room temperature overnight. It was then washedwith water and saturated brine solution, dried (Na₂SO₄) and thenconcentrated. The residue was recrystallized from ethyl acetate/hexaneto give 0.22 g (81% yield) of the desired compound as a white solid, mp:144-146° C. ¹H NMR (300 MHz, d₆-DMSO): δ2.01 [s, 3H, —CH₃], 4.33 [s, 6H,—CH₂Cl—], 6.75 [d, 2H, J=8.4 Hz, Ar—H], 6.99 [d, 2H, J=8.4 Hz, Ar—H].

1-(4-Acetoxy-phenyl)-1,1-bis-(4-methoxycarbonylmethoxyphenyl)ethane(I-14)

In step (i), methyl bromoacetate (0.95 mL, 10 mmol) was added dropwiseto a solution of 1,1,1-tris(4-hydroxyphenyl)ethane (1.53 g, 5 mmol),potassium carbonate (1.38 g, 10 mmol) and potassium iodide (16 mg, 0.1mmol) in 10 mL of acetone at room temperature under argon. The mixturewas refluxed for an additional 24 h, and then it was cooled to roomtemperature and extracted with ethyl acetate. The combined organic layerwas washed with 3% aqueous NaHCO₃, saturated brine solution, dried(Na₂SO₄) and then evaporated to dryness to afford a solid, which wasused without further purification in step (ii).

In step (ii), 0.45 g of the product from step (i) was refluxed with 5 mLof acetic anhydride for 3 h. The volatiles were removed under reducedpressure, and the residue was purified by flash chromatography elutingwith hexane-ethyl acetate (1:1) to give 0.43 g (88% yield) of thedesired compound as a colorless semisolid. ¹H-NMR (300 MHz, CDCl₃):δ1.57 [s, 3H, —CH₃], 2.28 [s, 3H, —COCH₃], 3.81 [s, 3H, —OCH₃], 4.61 [s,4H, —OCH₂—], 6.75-6.82 [m, 4H, Ar—H], 6.92-7.02 [m, 6H, Ar—H], 7.02-7.10[m, 2H, Ar—H].

1,1-Bis-(4-chloroacetoxyphenyl)-1-phenylethane (I-15)

Chloroacetyl chloride (0.08 mL, 1 mmol) was added dropwise to an icecooled solution of 1,1-bis(4-hydroxyphenyl)-1-phenylethane (0.14 g, 0.5mmol) and triethylamine (0.15 mL, 1 mmol) in 10 mL of CH₂Cl₂. Themixture was allowed to stand at room temperature overnight. It was thenwashed with water and a saturated brine solution, dried (Na₂SO₄) andthen concentrated. The residue was recrystallized from ethylacetate/hexane to give 0.2 g (91% yield) of the desired compound as awhite solid, mp: 97-98° C. ¹H NMR (300 MHz, d₆-DMSO): δ2.13 [s, 3H,—CH₃], 4.66 [s, 4H, —CH₂Cl—], 7.01-7.12 [m, 11H, Ar—H], 7.17-7.23 [m,1H, Ar—H], 7.25-7.33 [m, 1H, Ar—H].

2-Hydroxy-1,1-bis-hydroxymethyl-ethyl-ammonium;2-(3,6-dihydroxy-9H-xanthen-9-yl)-benzoate salt of II-5

Tris(hydroxymethyl)aminomethane (12 mg, 0.1 mmol) was added to asolution of 2-(3,6-dihydroxy-9H-xanthen-9-yl)-benzoic acid [33 mg, 0.1mmol, Cui, Y. et al., Yaoxue Tongbao, 1982, 17(9), 528] in 5 mL ofethanol and the mixture was stirred for an additional 2 h at roomtemperature. The mixture was evaporated to dryness under reducedpressure to give 37 mg (100% yield) of the desired compound as an orangecolored solid, mp>300° C. ¹H NMR (300 MHz, D₂O): δ3.75 [s, 6H, —CH₂—],5.76 [s, 1H, —CH], 6.94-6.99 [m, 1H, Ar—H], 7.09-7.13 [m, 2H, Ar—H],7.26-7.31 [m, 6H, Ar—H], 7.49-7.54 [m, 1H, Ar—H].

2-(3,6-Dichloro-9H-xanthen-9-yl)-benzamide (II-6)

A mixture of 2-(3,6-dichloro-9H-xanthen-9-yl)-benzoic acid (37 mg, 0.1mmol, Gronowska, J. and Dabkowska-Naskret, H. Polish J. Chem., 1981,55(10), 2151), thionyl chloride (0.5 mL) and toluene (5 mL) was refluxedfor 3 h under argon and concentrated under reduced pressure. Residualthionyl chloride was removed from the crude product mixture bycoevaporation with dry CHCl₃ (5 mL). Concentrated aqueous NH₃ (5 mL) wasthen added and the mixture stirred overnight at room temperature. Theprecipitate was filtered, and washed with H₂O. The crude product wasrecrystallized from ethanol to give 33 mg (90% yield) of the desiredcompound as a white solid, mp 218-220° C. ¹H NMR (300 MHz, d₆-DMSO):δ5.79 [s, 1H, —CH], 6.81-6.86 [m, 1H, Ar—H], 7.04-7.11 [m, 2H, Ar—H],7.16-7.28 [m, 6H, Ar—H], 7.39-7.45 [m, 1H, Ar—H], 7.76 and 8.23 [s, 2H,NH₂].

Potassium 2-[bis-(4-hydroxyphenyl)-methyl]-benzoate salt of II-7

Aqueous potassium hydroxide (1 mL of 0.1 N, 0.1 mmol) was added to asolution of 2-(bis-(4-hydroxyphenyl)-methyl)-benzoic acid [32 mg, 0.1mmol, Adamczyk, M. and Grote, J. Organic Preparations and ProceduresInternational, 2001, 33(1), 95] in 5 mL of ethanol and the mixture wasstirred for additional 2 h at room temperature. The mixture wasevaporated to dryness under reduced pressure to give 36 mg (100% yield)of the desired compound as a white solid, mp>300° C. ¹H NMR (D₂O, 300MHz): δ6.41 [s, 1H, —CH], 6.66 [d, 4H, J=8.8 Hz, Ar—H], 6.80 [d, 4H,J=8.8 Hz, Ar—H], 7.03 [d, 1H, J=7.7 Hz, Ar—H], 7.35 [t, 1H, J=7.7 Hz,Ar—H], 7.44 [t, 1H, J=7.7 Hz, Ar—H], 7.74 [dd, 1H, J₁=7.7 Hz, J₂=2.2 Hz,Ar—H].

Potassium 2-(3,6-dihydroxy-9H-xanthen-9-yl)-benzoate salt of II-8

Aqueous potassium hydroxide (1 mL of 0.1 N, 0.1 mmol) was added to asolution of 2-(3,6-dihydroxy-9H-xanthen-9-yl)-benzoic acid [33 mg, 0.1mmol, Cui, Y. et al., Yaoxue Tongbao, 1982, 17(9), 528] in 5 mL ofethanol and the mixture was stirred for additional 2 h at roomtemperature. The mixture was evaporated to dryness under reducedpressure to give 37 mg (100% yield) of the desired compound as a orangesolid, mp>300° C. ¹H NMR (D₂O, 300 MHz): δ5.76 [s, H, —CH], 6.94-6.99[m, 1H, Ar—H], 7.09-7.13 [m, 2H, Ar—H], 7.26-7.31 [m, 6H, Ar—H],7.49-7.54 [m, 1H, Ar—H].

N-(4,4′-Dihydroxybenzhydrylidene)-N′-(3-nitrophenyl)hydrazine (III-1)

A solution of 4,4′-dihydroxybenzophenone (0.32 g, 1.5 mmol) in 10 mL ofmethanol was added to a solution of 3-nitrophenylhydrazine hydrochloride(0.43 g, 2.25 mmol), concentrated sulfuric acid (0.3 mL) in 10 mL ofmethanol at 50° C. It was stirred at 50° C. for additional 2 h. Thereaction mixture was concentrated and diluted with 20 mL of water. Theprecipitates were separated by filtration and washed with 3% aqueousNaHCO₃ and water. The crude product was recrystallized from ethanol togive 0.4 g (77% yield) of the desired compound as a yellow solid, mp160-162° C. ¹H NMR (300 MHz, d₆-DMSO): δ6.71 [d, 2H, J=8.8 Hz, Ar—H],6.91 [d, 2H, J=8.1 Hz, Ar—H], 7.07 [d, 2H, J=8.8 Hz, Ar—H], 7.27 [d, 2H,J=8.8 Hz, Ar—H], 7.40 [dd, 1H, J=J₂=8.1 Hz, Ar—H], 7.49 [d, 1H, J=8.1Hz, Ar—H], 7.61 (dd, 1H, J₁=8.4 Hz, J₂=2.2 Hz, Ar—H], 7.99 [d, 1H, J=2.2Hz, Ar—H], 9.25 [s, 1H, NH], 9.78 and 9.62 [s, 1H, OH].

4,4′-Diacetoxybenzophenone-3-nitrophenylhydrazone (III-2)

Acetyl chloride (32 μL, 0.41 mmol) was added to an ice cold solution ofN-(4,4′-dihydroxybenzhydrylidene)-N′-(3-nitrophenyl)hydrazine (70 mg,0.2 mol) and triethylamine (60 μL, 0.41 mmol) in 5 mL of CH₂Cl₂. Themixture was then allowed to stand at room temperature overnight. It wasthen washed with water and a saturated brine solution, dried (Na₂SO₄)and then concentrated. The residue was recrystallized from ethylacetate/hexane to give 76 mg (88% yield) of the desired compound asyellow needles, mp: 88-90° C. ¹H NMR (300 MHz, d₆-DMSO): δ2.24 and 2.30[s, 3H, —CH₃], 7.07-7.15 [m, 2H, Ar—H], 7.31-7.40 [m, 4H, Ar—H],7.42-7.50 [m, 3H, Ar—H], 7.52-7.72 [m, 2H, Ar—H], 7.97-8.05 [m, 1H,Ar—H], 9.56 [s, 1H, NH].

N-(4,4′-Dichlorobenzhydrylidene)-N′-(3-nitrophenyl)hydrazine (III-3)

A solution of 4,4′-dichlorobenzophenone (0.38 g, 1.5 mmol) was added toa solution of 3-nitrophenylhydrazine hydrochloride (0.43 g, 2.25 mmol),concentrated sulfuric acid (0.3 mL) in 10 mL of methanol at 50° C. Itwas stirred at 50° C. for additional 2 h. The reaction mixture wasconcentrated to ¼ of its original volume and diluted with 20 mL ofwater. The precipitates were separated by filtration and washed with 3%aqueous NaHCO₃ and water. The crude product was recrystallized fromethanol to give 0.46 g (81% yield) of the desired compound as a yellowneedle, mp 170-172° C. ¹H NMR (300 MHz, d₆-DMSO): δ7.35 [d, 2H, J=7.7Hz, Ar—H], 7.37-7.49 [m, 5H, Ar—H], 7.58 [d, 1H, J=8.4 Hz, Ar—H], 7.64[d, 2H, J=8.4 Hz, Ar—H], 7.69 [d, 1H, J=8.4 Hz, Ar—H], 8.02 [s, 1H,Ar—H], 9.63 [s, 1H, NH].

The following compounds can be prepared according to the indicatedreferences (all of which are incorporated by reference):

1,1,1-Tris-(4-dimethylaminophenyl)methanol (I-1)

-   -   Lohmann, G. Y., U.S. Pat. No. 3,689,495.

1,1,1-Tris-(4-chloroacetoxyphenyl)ethane (I-2)

-   -   Mott, G. N. and Johnson, T. S. European Patent No. Publication        No. 475628.

1,1,1-Tris-(4-methoxycarbonylmethoxyphenyl)ethane (I-4)

-   -   Hayakawa, T., et al., Polymer J., 2000, 32(9), 784.

Sodium 1,1,1-tris-(4-methoxyphenyl)acetate (I-5)

-   -   Ford-Moore A. H., J. Chem. Soc., 1962, 1445.

1,1,1-Tris-(4-methoxyphenyl)methanol (I-6)

-   -   Nixon, A. C. et al., J. Am. Chem. Soc., 1955, 77(11), 3044.

4,4′,4″-Trimethoxytrityl tetrafluoroborate (I-7)

-   -   Henderson, A. P., et al., J. Chem. Soc. Perkin Trans. 1, 1997,        3407.

1,1,1-Tris-(4-methoxyphenyl)acetic acid (I-8)

-   -   Brain, E. G. et al., J. Chem. Soc., 1962, 1445.

1,1,1-Tris-(4-acetoxyphenyl)ethane (I-9)

-   -   Vicari, R. and Bodman, M. P., U.S. Pat. No. 5,362,843.

1,1-Bis-(4-acetoxyphenyl)-1-phenyl-ethane (I-10)

-   -   McGreal, M. E. et al., J. Am. Chem. Soc., 1939, 61, 345.

3,3-Bis[4-(acetyloxy)phenyl]-1,3-dihydro-2H-indol-2-one (I-16)

-   -   Preiswerk, E., U.S. Pat. No. 1,624,675.

1,1-Bis(4-dimethylaminophenyl)-1-phenyl-methanol (I-17)

-   -   Gilman, H. and Jones, R. G., J. Am. Chem. Soc., 1940, 62, 1243.

1,1-Bis(4-amino-3,5-dimethylphenyl)-1-(4-hydroxyphenyl)-ethane (I-18)

This compound is available from Specs (Netherlands, Cat. No.AG-205/32370012).

3′,6′-Dichlorofluorescein (II-1)

-   -   Deno, N. C. and Evans, W. L., J. Am. Chem. Soc., 1957, 79, 5804.

3′,6′-Fluorescein diacetate (II-2)

-   -   Hurd, C. D. and Schmerling, L., J. Am. Chem. Soc., 1937, 59,        112.

2-(3,6-Dichloro-9H-xanthen-9-yl)-benzoic acid (II-3)

-   -   Gronowska, J. and Dabkowska-Naskret, H. Polish J. Chem., 1981,        55(10), 2151.

2-(3,6-Dihydroxy-9H-xanthen-9-yl)-benzoic acid (II-4)

-   -   Cui, Y. et al., Yaoxue Tongbao, 1982, 17(9), 528

2′,7′-Dichlorofluorescein diacetate (II-9)

-   -   Brandt, R. and Keston, A. S., Anal. Biochem., 1965, 11(1), 6.        Wild Type and Mutant Human TNF Receptor 1 Cloning, Expression        and Purification

The ectodomain of wild type TNF receptor 1 was obtained by PCR frompKP13 (as described in Beutler et al., Annu. Rev. Biochem. 57, 505-518(1988)) with 5′ primer AAA AAA CAT ATG TAC CCC TCA GGG GTT ATT GG (SEQID NO:2) and 3′ primer CCG CTC GAG TCA ATG ATG ATG ATGATG ATG TGT GGTGCC TGA GTC CTC AG, (SEQ ID NO:3) and constructed into PET21 (Novagen,San Diego, Calif.) between Nde1 and XhoT, verified by sequencing. MutantTNF receptor 1 was obtained from site-directed mutagenesis by using aQuikChange mutagenesis kit (Stratagene, La Jolla, Calif.). The plasmidwas then transformed into Origami™ (DE3) (Novagen, San Diego, Calif.).The cells were grown until A600 was 0.6 and were induced by addition of0.2 mM of IPTG. The cells were then induced for 3 h and harvested bycentrifugation at 3500 rpm for 10 min. The wild type and mutant TNF-R1were all expressed in the inclusion bodies of the cells and wereextracted and refolded as described in Lin et al., Biotechniques 11, 748(December 1991). Briefly, the cell pellets from 100 mL culture wereresponded in 5 mL ice-cold buffer A (20 mM Tris-HCl, pH 7.5, 20%Sucrose, 1 mM EDTA) for 10 min, centrifuged at 6000 rpm for 5 min at 4°C., then re-suspended in 50 mL of ice-cold water for 10 min, andcentrifuged at 8200 rpm for 5 min at 4° C. The pellet was suspended in10 mL of Buffer P (PBS containing 5 mM EDTA, 1 mM PMSF, 0.1% Aprotinin)and sonicated. After sonication, the cell suspension were incubated withDNase I (400 μg/10 mL) for 10 min at room temperature. The suspensionwas further diluted by adding 40 mL of Buffer P and centrifuged at11,000 rpm for 30 min, 4° C. The pellet was then washed twice withBuffer W (PBS containing 25% Sucrose, 5 mM EDTA, 1% Triton) for 10 minat 4° C. and centrifuged at 15,000 rpm for 10 min, 4° C. The pellet wasthen resuspended in 10 mL of Buffer U2 (50 mM Tris-Hcl, pH 8.0, 8 MUrea) on ice for 1 h, centrifuged at 11,000 rpm for 30 min at 4° C. Thesupernatant was added to 1 L of Buffer R (50 mM Tris-HCl, pH8.0, 20%glycerol, 1 mM PMSF, 0.1% Aprotinin) for refolding, stirred gentlyovernight at 4° C. to renature the protein.

The refolded protein solution was centrifuged at 11,000 rpm for 30 minat 4° C. to remove the aggregation. The supernatant was mixed with Talonmetal affinity resin (QIAexpressonst™, Qiagen, Inc, Valencia, Calif.),rocked for 2 h at 4° C., and then washed three times with 50 mM NaH₂PO₄containing 300 mM NaCl and 20 mM imidazole. The purified TNF-R1 wasfinally eluted with 51 mM NaH₂PO₄ containing 300 mM NaCl and 150 mMimidazole, pH 8.0.

Commercially obtained TNF receptor 1 may also be used for any of theexamples disclosed herein.

Binding of Allosteric Inhibitor Leads to Perturbation of w107

Compound I-9 was tested for its ability to bind to an isolated andpurified TNF-R1. Isothermal titration calorimetry (ITC) was employed todeduce the binding characteristics and the results are shown in FIG. 1.I-9 bound selectively to TNF-R1 at one site with an affinity of 2.2×10⁻⁶M⁻¹.

It appears that there are no large detectable conformational changes onligand binding (Banner et al., Cell 73, 431 (1993)), so it is thoughtthat conformational alterations may be subtle, perhaps on the order of 2Å (0.2 nm). Fluorescence quenching can identify small modulating changesin proteins. Only one tryptophan residue exists in the TNF-R1 ectodomainand it is located in the WP9 loop.

The results from fluorescence quenching induced by acrylamide followingbinding of compound I-9 are shown in FIG. 2. The residue W107 in the WP9loop fluoresces around 340 nm. In this set of experiments, the resultantconcentration of quencher ranged up to 0.25 M, quenching 77.4% of thetotal intrinsic fluorescence of TNF-R1. The Stem-Volmer constant forTNF-R1 quenching by acrylamide calculated from the slope of the plot is14.4±0.2 M⁻¹, compared to 11.6±0.2 M⁻¹ for TNF-R1 in the presence of thetest compound, indicating that binding of I-9 to TNF-R1 introducedconformational changes in the TNF-R1 which partly protects W107 from thequencher. Thus binding of I-9 to the receptor changed the disposition oftryptophan-107.

Binding of Allosteric Inhibitor to Mutant TNF-R1

Mutations of the TNF-R1 receptor were made at residues 82Q and 112F inthe cavity, which were mutated to 82E and 142E respectively. Thestructural integrity of the mutant receptor was verified by ligandbinding in surface plasmon resonance (SPR) studies. Ligand, TNF-α, boundto the wild type TNF-R1 (k_(d)=3.79×10⁻¹⁰ M) and with mutant TNF-R1(k_(d)=4.65×10⁻⁶ M) suggesting that the mutations affected the ligandbinding sites to some extent. Using ITC, it was found that compound I-9no longer bound to the mutant receptor (FIG. 1B). TNF-α retained theability to bind to the mutant receptor, albeit with somewhat reducedaffinity. These studies confirm that the test compound bound to a singleand specific cavity on wild type TNF-R1.

Inhibition of TNF-α-Mediated Cytotoxicity

L929 cultured murine fibroblasts cells were obtained from American TypeCulture Collection (Manassas, Va.). Tissue culture reagents were fromInvitrogen or Sigma-Aldrich. TNF-α and Actinomycin D were fromSigma-Aldrich. Alamar Blue reagents (Cell Titer Blue™) were fromPromega. The test compounds I-1 through I-16, II-1 through II-8, andIII-1 through III-3 were obtained commercially or can be prepared asdescribed above. The test compounds were individually dissolved indimethylsulfoxide (DMSO). The test compounds were maintained at 4° C.when not in use. Other reagents were high-purity (ACS-grade, HPLC-grade,MilliQ water, or similar).

Stock L929 cells were grown on tissue culture plastic in complete DMEM(Dulbecco's Modified Eagles Medium) supplemented with 10% FBS (FetalBovine Serum), NEAA (non-essential amino acids), and glutamine. L929cells were plated using the same medium on 96-well tissue culture platesat high density (i.e., ˜4×10⁴ cells/well, or similar) before use.

Approximately 20 h after plating, the L929 cells on 96-well plates werere-fed with fresh medium containing one of the test compounds in aconcentration selected from 100 μM, 50 μM, 25 μM as a “pre-treatment.”Approximately 30-60 min later, samples were treated with an additionalamount of test compound (in the same amount and at the sameconcentration as the pre-treatment) prepared in medium containing TNF-α(200 pg/mL), and actinomycin D (2 μM). The final concentration of TNF-αin assays was 100 pg/mL. The final concentration of actinomycin D inassays was 1 μM. The plates were incubated for an additional 22-23 h,the Alamar Blue assay reagents were added and metabolic cell viabilitywas determined from reduction of a fluorogenic Alamar Blue derivative.

The temperature was maintained at 37° C. with 5% CO₂ and humidified. Themetabolic viability was measured 1-2 h after addition of assayreagents/Alamar Blue derivative using TECAN SaFire fluorescence platereader (Tecan Group Ltd., Maennedorf, Switzerland).

Inhibition of TNF-α induced cytolysis by a test compound (X) at a givenconcentration (y) was calculated as follows:

${\%\mspace{14mu}{Inhibition}} = {100*\left\{ \frac{{{Viability}\left( {{X\; y\;\mu\; M},{{{ActD}\&}{TNF}}} \right)} - {{Viability}\left( {{{ActD}\&}{TNF}} \right)}}{{{Viability}({ActD})} - {{Viability}\left( {{{ActD}\&}{TNF}} \right)}} \right\}}$

Results for compounds tested in the TNF-α mediated cytolysis assay aregiven in Table 1.

TABLE 1 Inhibition of TNF-α Mediated Cytolysis of L929 Cells %Inhibition at: Compound 100 μM 50 μM 25 μM I-1 40.9 63.3 44.0 I-2 18.157.4 24.9 I-3 12.4 17.7 23.0 I-4 −11.6 27.9 18.4 I-5 12.1 5.0 1.7 I-632.5 25.9 12.0 I-7 25.9 30.2 15.1 I-8 18.4 6.9 2.7 I-9 50.9 65.2 37.2I-10 71.9 46.6 20.7 I-11 14.8 36.7 13.9 I-12 14.2 16.9 14.5 I-13 38.972.2 35.4 I-14 45.3 30.2 13.4 I-15 −19.8 73.8 29.7 I-16 17.1 11.5 4.3I-17 −17.9 −17.0 −16.0 I-18 53.8 36.6 22.6 II-1 7.6 8.8 20.7 II-2 26.023.5 25.4 II-3 52.4 45.6 36.2 II-4 23.2 23.6 33.3 II-5 21.5 16.3 25.1II-6 −21.7 −6.1 43.3 II-7 13.2 19.1 22.5 II-8 25.6 24.7 23.6 II-9 22.010.5 6.3 III-1 −20.3 42.5 33.2 III-2 −19.8 42.0 26.9 III-3 8.1 3.4 6.6Inhibition of TNF-α Signaling in L929 Cells

The effect of compound I-9 on TNF-α signaling was examined in L929 cells(American Type Culture Collection, Manassas, Va.). Cells were culturedin RPMI (Invitrogen) containing 5% fetal bovine serum. Cells(1×10⁶/well) were cultured in 6-well plates for 12 h, treated with orwithout small molecule for 2 h, and then stimulated with TNF-α at 20ng/mL for the indicated periods. Cells were then washed with ice-coldphosphate-buffered saline and lysed with lysis buffer. Cell lysates(15-30 μg) were separated by 12% SDS-PAGE, electroblotted ontonitrocellulose membrane (Osmonics, Westborough, Mass.), and probed withanti-phospho-IκBα, anti-IκBα, anti-phospho-p38, anti-p38, andanti-β-actin antibodies (Cell Signaling Technology, Inc., Beverly,Mass.) and developed using an enhanced chemiluminescence (ECL) system(Amersham Biosciences, Piscataway, N.J.). Results showed that treatmentof L929 cells with compound I-9 reduced production of phospho-IκBα andphospho-p38 following TNF-α treatment (FIG. 3A).

Inhibition of TNF-α Signaling in THIP1 Cells

THP1 cells (Human acute monocytic leukemia cell line, American TypeCulture Collection, Manassas, Va.) were cultured in RPMI with thesupplement of 50 mM HEPES, 1 mM Na Pyruvate, 50 μM of 2-ME, 2.5 mg/mL ofglucose, 50 μg/mL of gentamicin and 10% of FBS. Cells were cultured in12-well plate at a density of 6×10⁵/well with supplement of 100 ng/mLPMA. Cells were plated 72 hrs to differentiate. Cells were thenpretreated with or without small molecule for 2 hr, followed bytreatment with TNF-α at 20 ng/mL at the indicated periods. Cells werethen lysed and analyzed by western blotting in the same manner asdescribed above for L929 cells. Results showed that treatment withcompound I-9 inhibited TNF-α mediated I-kBα and p38 phosphorylation inTHP1 cells, as shown in FIG. 3C.

Effect on EGF-Signaling in NE91 Cells

To verify the effects of I-9 were specific to TNF-α signaling, theeffect of I-9 on EGF-signaling was tested in NE91 cells (American TypeCulture Collection, Manassas, Va.). NE91 cells were cultured in RPMImedium containing 10% FBS in 6-well plate at the density of 1×10⁶/wellfor 12 hr, followed by 2 hr treatment with or without I-9. Cells werethen stimulated with EGF at 100 ng/mL for the indicated time. Cells werethen lysed and analyzed by western blotting in the same manner asdescribed above for analysis of TNF-α signaling in L929 cells. Resultsshowed that compound I-9 failed to induce changes in EGF-inducedsignaling (FIG. 3B), indicating that I-9 specifically altered the TNF-R1signaling pathways.

Inhibition of Collagen Induced Arthritis

Activity of compound I-9 was studied in a mouse collagen inducedarthritis system, which is a model for human rheumatoid arthritis. Sixto eight weeks old male DBA/1 mice were immunized by multipleintradermal injections of 100 μg chicken type II collagen (SigmaChemical Co., St. Louis, Mo.) in 100 μl of 0.1 M acetic acid emulsifiedin an equal volume of complete Freund's adjuvant and were thenchallenged with the same antigen preparation i.p. on the 21st day.Animals were injected daily with the compound I-9 at different dosages(2-4 mg/kg/day) beginning on day 21 and animals were examined physicallyevery other day in a blinded manner.

In this model, disease typically develops 7-10 days after the secondimmunization, and the severity of disease can be determined by physicalexamination, joint histochemistry, or both techniques. Mice treated withcompound I-9 showed a dose dependent decrease in the clinical symptomsof arthritis compared with untreated or control groups (FIG. 4).Histological analysis of ankle joints of the animals revealed that thetreated mice have less synovial tissue and reduced matrix proteoglycans.Infiltration was markedly reduced and matrix proteoglycans were notdepleted. Cartilage destruction was also prevented in the I-9 treatedgroup.

Effect of TNF Receptor 1 Dependent In Vivo Collagen Induced Arthritis

Male DBA/1 mice (6-8-wk-old) were purchased from Jackson Laboratory (BarHarbor, Me.) and housed in University of Pennsylvania Animal CareFacilities. Animals were maintained in accordance with guidelines ofInstitutional Animal Care and Use Committee (IACUC) of the University ofPennsylvania. For CIA induction, mice were immunized by multipleintradermal injections of 100 μg chicken type II collagen (SigmaChemical Co., St. Louis, Mo.) in 100 μL of 0.1M acetic acid emulsifiedin an equal volume of complete Freund's adjuvant. Mice were challengedwith the same antigen preparation i.p. at the 21st day. Mice wereinjected daily with the test compound at different dosage (2 and 4 mg/kgof body weight) from day 21. Disease develops 7-10 day after the secondimmunization. Mice were examined physically every other day in a blindmanner. Their paws were scored individually as follows: 0=normal;1=Erythema and mild swelling confined to the ankle joint or toes;2=Erythema and mild swelling extending from the ankle to the midfoot orankle joint; 3=Erythema and moderate swelling extending from the ankleto the metatarsal joints; and 4=Erythema and severe swelling encompassthe ankle, foot, and digits. The maximum disease score per foot is 4,and the maximum disease score per mouse is 16. For histologicalexamination of the joint, mice were killed at different time points, andtheir paws were collected and fixed in 10% formalin. The paws were thendecalcified in hydrochloric acid, embedded in paraffin, sectioned, andstained with hematoxylin and eosin (H&E).

Other Methods

Fluorescence Quenching Studies

Quenching experiments with acrylamide were performed in stirred cells,at 25° C., titrating from a stock of 1 M acrylamide by adding 2.5 μl ofacrylamide each time. Recombinant TNF-R1 were at the concentration of 5μM in 1% DMSO. Tryptophan emission, monitored at 340 nm, was observedusing 295 nm excitation. Intensity data following quencher additionswere averaged over a 10-sec collection and were corrected for backgroundemission (paired control lacking of protein). Intensities, F, at givenquencher concentration, [Q], were analyzed using the Stem-Volmerequation,F ₀ /F=1+K _(S.V) ·[Q]

-   -   Where F₀ is the emission intensity of the protein in the absence        of quencher, and K_(S.V) is the Stem-Volmer constant for        quenching, given by the slope when data are plotted as F_(o)/F        versus [Q].

For synthesized small molecules, a test compound at 20 μM waspre-incubated with 5 μM TNF-R1 in 1% DMSO for 30 min, and titrated with1 M acrylamide the same way as TNF-R1 alone. The F₀/F was analyzed usingthe Stem-Volmer equation, and two slopes from TNF receptor and from TNFreceptor with the test compound were compared.

Kinetic Binding Studies by Surface Plasmon Resonance

Recombinant TNF receptor wild type and mutant were immobilized to theCM5 sensor chip with a surface density of 2,000 resonance units. Thebinding affinity of TNF-α to TNF-R1 was estimated by BiaCORE 3000(BiaCORE, Uppsala, Sweden) at 25° C. The apparent rate constants (k_(on)and k_(off)) and the equilibrium binding constant (K_(d)) for TNF/TNF-Rbinding interaction were estimated from the kinetic analysis ofsensorgrams, using the BIA evaluation 3.0 software (BiaCORE).

Isothermal Titration Calorimetry

The binding thermodynamics of inhibitors to the TNF receptor wasmeasured by isothermal titration calorimetry (ITC) using ahigh-precision VP-ITC titration calorimetric system (MicroCal Inc,Northampton, Mass.). The calorimetric cell containing wild type ormutant TNF receptor at a concentration of about 1 to 6 μM dissolved in 5mM Tris, pH 8.0 with 2% DMSO, was titrated with the inhibitors dissolvedin the same buffer. The concentration of inhibitor was 50-120 μM,depending on the solubility in buffer. Injection volumes were 10 μL. Allsolutions were properly degassed to avoid any formation of bubbles inthe calorimeter during stirring. The heat evolved upon each injection ofinhibitor was obtained from the integral of the calorimetric signal. Theheat associated with the binding of the inhibitor to TNF receptor wasobtained by subtracting the heat of dilution from the heat of reaction.The measurements were made at 25° C. Data were analyzed and fitted usingthe data analysis software supplied by MicroCal (Origin version 5.0).

* * *

The above specification, examples and data provide a description of themanufacture and use of the composition of the invention. Since manyembodiments of the invention can be made without departing from thespirit and scope of the invention, the invention also resides in theclaims hereinafter appended.

All references, including publications, patents, and patentapplications, cited herein are incorporated herein by reference.

1. A method of inhibiting tumor necrosis factor action, comprisingtreating a patient in need of such inhibition with an effective amountof a compound having a structure:

or a pharmaceutically acceptable salt thereof.
 2. A method of treatingrheumatoid arthritis, psoriasis, ankylosing spondylitis, Crohn'sdisease, or ulcerative colitis comprising administering to a patient inneed of such treatment an effective amount of a pharmaceuticalcomposition comprising an excipient and a compound having a structure:

or a pharmaceutically acceptable salt thereof.
 3. The method of claim 2,wherein the pharmaceutical composition is in the form of a capsule ortablet.
 4. The method of claim 3, wherein the capsule or tablet containsthe compound having a structure of formula (I) in an amount in the rangeof about 25 mg to about 200 mg.